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Molecular analysis of superoxide dismutase in Campylobacter lari

Molecular analysis of superoxide dismutase in Campylobacter lari Ann Microbiol (2014) 64:1347–1356 DOI 10.1007/s13213-013-0778-7 ORIGINAL ARTICLE Molecular analysis of superoxide dismutase in Campylobacter lari Takuya Nakajima & Takashi Kuribayashi & Shizuo Yamamoto & John E. Moore & Beverley C. Millar & Motoo Matsuda Received: 29 July 2013 /Accepted: 18 November 2013 /Published online: 12 December 2013 Springer-Verlag Berlin Heidelberg and the University of Milan 2013 Abstract The superoxide dismutase (SOD) gene clusters, segments with 28 C. lari isolates, including 14 UPTC isolates, sodB and sodC, and their adjacent genetic loci from a urease- gave positive results. C. lari organisms were shown to carry positive thermophilic Campylobacter (UPTC) CF89-12 strain both the sodB and sodC genes with extremely high frequency. were analyzed molecularly, and compared with those of ther- More high-SOD activity was seen with the C. lari isolates (n = mophilic campylobacters. The UPTC CF89-12 strain carried 9), including UPTC, than was seen with the other three ther- sodB [structural gene 654 base pairs (bp)] and sodC (540 bp) mophilic Campylobacter and Helicobacter pylori organisms. genes, as did the Campylobacter lari RM2100 reference strain. . . However, the other three thermophilic Campylobacter jejuni, Keywords Oxidative stress defense SOD Thermophilic . . C. coli and C. upsaliensis reference strains carried only a single campylobacters C. lari Urease-positive thermophilic sodB gene, and no sodC. Although sodB and sodC in the Campylobacter UPTC strain shared relatively high nucleotide sequence simi- larities (92.9%and91.7%, respectively) with the correspond- ing genes in the C. lari RM2100 strain, the sodB gene in the Introduction UPTC CF89-12 and C. lari RM2100 strains shared relatively low nucleotide sequence similarities with those in C. jejuni Oxidative stress is an important matter for organisms NCTC11168 (80.8 % and 81.7 %), C. coli RM2228 (82.0 % employing oxygen as a terminal electron acceptor, since the and83.1%)and C. upsaliensis RM3195 (75.9 % and 77.0 %), combination of oxygen and an electron can generate reactive respectively. All PCR amplifications of sodB and sodC gene oxygen such as the superoxide (O ), the hydroxyl radical (HO⋅) and hydrogen peroxide (H O ) (Storz and Imaly 1999). 2 2 These reactive oxygen species can lead to damage of proteins, T. Nakajima M. Matsuda (*) nucleic acids and membranes (Atack et al. 2008). Reactive Laboratory of Molecular Biology, Graduate School of Environmental oxygen species are also generated by the immune system to Health Sciences, Azabu University, Sagamihara 252-5201, Japan kill invading microbes (Atack et al. 2008). Therefore, for e-mail: matsuda@azabu-u.ac.jp bacterial pathogens to survive, they must resist the reactive T. Kuribayashi S. Yamamoto oxygen stress encountered in both the host and the environ- Laboratory of Immunology, Graduate School of Environmental ment. Recent studies suggest that bacteria contain a wide Health Sciences, Azabu University, Sagamihara 252-5201, Japan range of enzymes involved in oxidative stress (Atack and J. E. Moore B. C. Millar Kelly 2009). Superoxide dismutase (SOD) is considered im- Department of Bacteriology, Northern Ireland Public Health portant in protection of aerobes against oxidant damage (Scott Laboratory, Belfast City Hospital, Belfast BT9 7 AD, UK et al. 1987). Thermophilic Campylobacter jejuni and Campylobacter J. E. Moore School of Biomedical Sciences, University of Ulster, coli species are curved, Gram-negative organisms, and are Londonderry BT52 1SA, Northern Ireland, UK the most commonly recognized causes of acute bacterial diarrhea in the Western world (Skirrow and Benjamin 1980; J. E. Moore Benjamin et al. 1983; Blaser et al. 1983). Thermophilic Centre for Infection and Immunity, Queen’s University, Belfast BT9 7AB, Northern Ireland, UK Campylobacter lari species was first isolated particularly 1348 Ann Microbiol (2014) 64:1347–1356 Table 1 Thermophilic Campylobacter isolates analyzed in the present from seagulls (Skirrow and Benjamin 1980; Benjamin et al study and accession numbers of nucleotide sequence data of sod genes 1983). C. lari has also been shown to occasionally be a cause clusters accessible in the DDBJ/EMBL/GenBank of clinical infection (Nachamkin et al. 1984;Martinot etal. Campylobacter Source Country Accession no. 2001; Werno et al. 2002). In addition, an atypical group of urease-positive thermophilic Campylobacter (UPTC) organ- UPTC CF89-12 River water Japan AB736167 isms have been isolated from the natural environment in C. lari RM2100 Human United States NC_012039 England in 1985 (Bolton et al. 1985). Thereafter, these organ- C. jejuni NCTC11168 Human England NC_002163 isms were described as a biovar or variant of C. lari (Bolton C. coli RM2228 Chicken United States AAFL01000005 et al. 1985; Mégraud et al. 1988). Subsequent isolates were C. upsaliensis RM3195 Human United States AAFJ01000003 reported in Europe (Mégraud et al. 1988;Owen etal. 1988; Bezian et al. 1990; Wilson and Moore 1996;Kaneko etal. 1999; Endtz et al. 1997; Matsuda et al. 2003); and in Japan (Matsuda et al. 1996; Matsuda et al. 2002). Thus, at least these thermophilic Campylobacter reference strains analyzed in two representative taxa, namely urease-negative (UN) C. lari the present study and the accession numbers of the nucleotide and UPTC exist within the species C. lari (Matsuda and sequence data of their sodB and sodC genes clusters are also Moore 2004). shown in Table 1. Regarding the oxidative stress defense system in Campylobacter cells were cultured on Mueller-Hinton Campylobacter, van Vlietetal. (2002) reviewed the role of agar (Oxoid, Hampshire, UK) containing defibrinated iron in Campylobacter gene regulation, metabolism and oxi- horse blood [7 % (v/v); Nippon Bio-test, Tokyo, Japan], supplemented with Butzler Campylobacter-selec- dative stress defense. In addition, Atack et al. (2008)sug- gested that thioredoxin-linked thiol peroxidase and tive medium (Virion, Zurich, Switzerland), under bacterioferritin comigratory protein are partially redundant microaerophilic conditions produced by BBL Campypak antioxidant enzymes that play an important role in protection Microaerophilic System Envelopes (Becton Dickinson, of C. jejuni against oxygen-induced oxidative stress. More Franklin Lakes, NJ) at 37 °C for 48 h. Cells were further recently, oxidative stress in C. jejuni, responses, resistance cultured on Mueller-Hinton agar under the same and regulation (Atack and Kelly 2009), characterization of the microaerophilic conditions. oxidative stress stimulon and peroxide-sensing regulator regulon of C. jejuni (Palyada et al. 2009) and regulation of the oxidative stress response by the Campylobacter oxidative Genomic DNA preparation stress regulator—an essential response regulator in C. jejuni Template DNA was prepared using sodium dodecyl sulfate (Hwang et al. 2011)—were reported. Regarding the oxidative stress defense system in C. lari, and proteinase K treatment, phenol-chloroform extraction and Miller et al. (2008) described recently sodB and sodC in UN ethanol precipitation (Harrington et al. 1997), and adjusted to C. lari RM2100 strain following analysis of the complete approximately 500 ng/μL. genome sequence in this organism. However, to our knowl- edge, the system has not yet been described in UPTC organ- isms. Therefore, the present study aimed to identify sod genes Construction of the genomic DNA library of the UPTC clusters in the Campylobacter lari organisms, including CF89-12 strain and nucleotide sequence determination UPTC isolates, and to perform genotypic and phenotypic TM comparisons of the SOD genes within thermophilic A genomic DNA library was constructed using NEBNext campylobacters. DNA Sample Prep. Reagent Set 1 (New England BioLabs, Japan, Tokyo, Japan). The DNA was fragmented using Covaris S-Series (Covaris, Woburn, MA) and separated by Materials and methods agarose gel electrophoresis [300–500 base pairs (bp)]. Cluster generation was carried out using the constructed library DNA Campylobacter lari isolates and growth conditions used as templates with Cluster Station and Cluster Generation Kit in the present study (Illumina, San Diego, CA). The nucleotide sequence (sequence reads 75 bp) was de- The Japanese strain, UPTC CF89-12 (Table 1), which was termined using Genome Analyzer IIx and Sequencing Kit isolated from the water of the Asahigawa River, Okayama (Illumina). De novo assembly of the paired-end reads prefecture, Japan (Matsuda et al. 1996), was employed for the (35 bp) was carried out using Edena (V2.1.1., http://www. construction of a genomic DNA library, cloning, sequencing genomic.ch/edena.php) and Velvet (V0.7.11, http://www.ebi. ac.uk/~zerbino/velvet/). and characterization of the sod genes clusters. Other Ann Microbiol (2014) 64:1347–1356 1349 f-ClsodB ATGTTTGAATTAAGAAAACTACCTTATGAAGCAGATGCTTTTGGAGACTTTTTAAGTGCAGAAACTTTTGCGTTTCATCATGGCAAACACCACCAAACTTATGTAAATAAC 50,450 C. lari RM2100 50,340 ..................T................C...........T........C...........C..A....................................... 3,885 UPTC CF89-12 3,375 C. jejuni NCTC11168 166,373 ..................T..........TA.CA..........T..T.....G.....T.........AGC.A.........A.....T...A.T........T.CA..T 166,483 C. coli RM2228 49,969 ..................T..........TA.CA..........T..T...........T.........AGT.A.........A.........A.T..........CA..T 50,079 110,491 C. upsaliensis RM3195 110,381 ..................T.......C..TA.TA..........C..T...........T..G..C...AGT.A......C.......................T.....T ****************** ******* ** * * ******** ** ***** ** ** ** ** ** * ****** ** ***** *** * ******** * ** r-ClsodB 50,993 C. lari RM2100 50,886 TACGCGCACATTAACTGGGAATTCGTAGCAAAAGCTTATGAATGGGCTATCAAAGAAGGTATGAACTCAGTAAGTTTTTATGCAAACGAATTGCACCCTGTAAAATAA .....A..T..............T..................................................C...............C.T..T..AC....C... 4,428 UPTC CF89-12 4,321 ..T..T..T..............T..T...........C.........T.A........C...GGA.....T..C...........T...C.T............... 167,035 C. jejuni NCTC11168 166,918 C. coli RM2228 50,524 ..T..T..T..............T........................T.A............GGA.....T..C.....C.........C.T..T............ 50,631 111,022 C. upsaliensis RM3195 110,915 .....T..T..........CT..T..T..............G......T.G.....G.......G......T..C.....C.....T...C.A........G...... ** ** ** ********** ** ** *********** ** ****** * ***** ** *** ***** ** ***** ***** *** * ** ** * ** *** f-ClsodC UNC. lari RM2100 106,256 ATGAAAAAAATAATTATAGGCTCTTTACTAGCATCAAGCTTTTTAATTGGGGCAAATTTAGAAAATTTTGATCCAAAAGCACAAAAAGATCATTTAGTTA 106,157 ...........T..AC.............G........T..C.......................................................... 1,981 UPTC CF89-12 1,882 *********** ** ************* ******** ** ********************************************************** r-ClsodC 105,717 UNC. lari RM2100 105,756 CGGTGGTGGCGCTAGAATGGCTTGTGGAGTTATTAAGTAA .........T.................G.A.TA.TCC... 2,421 UPTC CF89-12 2,382 ********* ***************** * * * *** Fig. 1 a Nucleotide sequence alignment analyses of the putative sodB of the sodB gene was omitted from the strains, respectively (nt 3,886– gene in the UN Campylobacter lari RM2100, C. lari UPTC CF89-12, C. 4,320 bp for UPTC CF89-12). b Nucleotide sequence alignment analyses jejuni NCTC11168, C. coli RM2228 and C. upsaliensis RM3195 strains. of the sodC gene in the UN C. lari RM2100 and UPTC CF89-12 strains. Numbers at the left and right refer to the nucleotide positions determined. A central region of approximately 400 bp (nt 1,982–2,381 bp for the Dots Identical bases (changes are explicit), dashes deletions; asterisks UPTC CF89-12) of the sodC gene was omitted identical positions in all cases. A central region of approximately 430 bp Putative sodB and sodC gene sequence analyses comparison of nucleotide and deduced amino acid sequence similarities with those of the corresponding Sequence analyses of the putative sodB and sodC genes and sodB and sodC genes from the UN C. lari RM2100 their adjacent genetic loci from the UPTC CF89-12 strain strain (DDBJ/ EMBL/GenBank accession no. were carried out using the GENETYX-Windows computer NC_012039). software (version 9; GENETYX, Tokyo, Japan). Nucleotide sequence alignment analyses of the puta- The putative sodB and sodC genes sequences from tive sodB and sodC from the UPTC CF89-12 strain the UPTC CF89-12 strain were identified based on were carried out with the accessible sequence data of Fig. 2 Schematic representations of the putative sodB (a)and sodC (b) genes and their adjacent genetic loci in the UPTC CF89-12 strain, showing the locations of 12 3 4 6 the primer pairs of f-/r-ClsodB f-ClsodB r-ClsodB and f-/r-ClsodC designed in silico for the sodB and sodC genes segments, and their sequences (c). The gene numbers (1–6) in the box correspond to those in Figs. 3a,c and 4a,c 12 3 4 5 f-ClsodC r-ClsodC Primer Nucleotide sequence (5'-3') CAAACACCACCAAACTTATG f-ClsodB r-ClsodB GCCCATTCATAAGCTTTTGC GGCTCTTTACTGGCATCAAG f-ClsodC ACCCCACAAGCCATTCTAGC r-ClsodC 1350 Ann Microbiol (2014) 64:1347–1356 a C. lari UPTC CF89-12 (AB736167) 12 3 4 6 b UN C. lari RM2100 (NC_012039) 12 3 4 5 6 UPTC CF89-12 C. lari RM2100 No. Gene Product 5' end 3' end 5' end 3' end 1 201 815 46,766 47,380 Cla_0047 conserved hypothetical protein (DUF285 domain protein) 2 948 3,227 47,512 49,791 nrdD anaerobic ribonucleoside triphosphate reductase 3 3,175 3,708 49,739 50,272 Cla_0049 anaerobic ribonucleoside triphosphate reductase activating protein 4 3,775 4,428 50,340 50,993 sodB superoxide dismutase (Fe) 5 - - 51,053 52,426 Cla_0051 putative C4-dicarboxylate transporter 6 5,448 4,459 53,438 52,449 purM phosphoribosylaminoimidazole synthetase d C. upsaliensis RM3195 (AAFJ01000003) 1326 4 5 C. upsaliensis RM3195 No. Gene Product 5' end 3' end 1 108,266 106,944 clpP ATP-dependent Clp protease, proteolytic subunit ClpP 2 108,371 108,955 folE GTP cyclohydrolase I 3 108,966 110,093 CUP1761 hypothetical protein 4 110,381 111,022 CUP1762 superoxide dismutase (sodB) 5 111,646 111,023 CUP1763 phosphoribosyl-ATP pyrophosphatase/phosphoribosyl-AMP cyclohydrolase 6 111,951 111,643 CUP1764 TfoX domain protein, putative f C. jejuni NCTC11168 (NC_002163) 12 5 6 7 8 c C. coli RM2228 (AAFL01000005) 15 34 7 8 C. jejuni NCTC11168 C. coli RM2228 No. Gene Product 5' end 3' end 5' end 3' end 1 165,580 165,017 48,624 48,061 NA putative integral membrane protein 2 166,105 165,938 - - NA putative periplasmic protein 3 - - 49,032 49,439 NA integral membrane protein, putative 4 - - 49,570 49,863 NA integral membrane protein, putative 5 166,373 167,035 49,969 50,631 sodB superoxide dismutase (fe) 6 167,050 167,794 - - NA hypothetical protein 7 167,807 169,012 51,863 50,658 NA putative saccharopine dehydrogenase 8 169,962 169,054 52,804 51,899 NA putative iron-uptake ABC transporter ATP-binding protein Fig. 3 Schematic representations of the putative sodB gene and its coli RM2228. Further details shown in c, e and h are 5′ end (np, bp) and adjacent genetic loci in strains a C. lari UPTC CF89-12, b UN C. lari 3′ end (np, bp); − absent, NA not available RM2100, d C. upsaliensis RM3195, f C. jejuni NCTC11168 and g C. other thermophilic Campylobacter strains, UN C. lari upsaliensis RM3195 (AAFJ01000003) using CLUSTAL W RM2100 (NC_012039), C. jejuni NCTC11168 software (1.7 program) (Thompson et al 1994), which is (NC_002163), C. coli RM2228 (AAFL01000005) and C. incorporated in the DDBJ. Ann Microbiol (2014) 64:1347–1356 1351 Fig. 4 Schematic C. lari UPTC CF89-12(AB736168) representations of the putative sodC gene and its adjacent genetic loci in a C. lari UPTC CF89-12 and b UN C. 12 3 4 5 lari RM2100strains. c Details shown are 5′ end (np, bp) and 3′ end (np, bp) UN C. lari RM2100 (NC_012039) 12 3 4 5 UPTC CF89-12 C. lari RM2100 No. Gene Product 5' end 3' end 5' end 3' end 1 837 1 108,139 107,303 Cla_0124 conserved hypothetical protein, probable ATP/GTP-binding protein 2 1,778 903 107,237 106,359 Cla_0123 conserved hypothetical protein, putative transcriptional regulator (AraC family) 3 1,882 2,421 105,717 106,256 sodC superoxide dismutase (Cu/Zn) 4 2,976 2,455 105,685 105,164 Cla_0121 hypothetical protein Cla_0121 5 3,609 3,019 105,120 104,527 Cla_0120 conserved hypothetical lipoprotein Primer design for sodB and sodC gene segments ufacturer’s instructions and the procedures described by Morishita et al. (2012). One unit was defined as the Two primer pairs (f-/r-ClsodB and f-/r-ClsodC) were designed SOD amountin20 μL of a sample exhibiting 50 % inhibition in silico for amplification of the sodB and sodC genes seg- of water-soluble tetrazolium salt (WST) reduction. SOD ac- ments based on sequence information for the UPTC CF89-12 tivities were divided into three groups of high, moderate and strain (AB736167 and AB736168) and the four other thermo- low activity, according to the description by Morishita et al. philic Campylobacter reference strains shown above, in order (2012). SOD activity was determined three times in indepen- to identify the sodB and sodC genes segments in the individ- dent experiments. ual C. lari isolates (n =14 for UPTC; n =14 for UN C. lari) (Figs. 1, 2). Nucleotide sequence alignment analyses to design the Results primer pairs was carried out using CLUSTAL W soft- ware (1.7 program) (Thompson et al. 1994), as de- Molecular identification of putative sodB and sodC genes scribed above. clusters and their adjacent genetic loci in the UPTC CF89-12 strain genomic DNA Superoxide dismutase activity determination During the process of genome sequence analysis of a repre- Superoxide dismutase (SOD) activity was determined sentative taxon of C. lari UPTC, sodB and sodC genes were using the SOD-Assay Kit-WST (Dojindo Molecular identified in the genomic DNA of environmental Japanese Technologies, Kumamoto, Japan) according to the man- UPTC CF89-12. sodB and sodC genes clusters and their Table 2 Sequence similarities (%) of the nucleotide (upper right) and amino acid (lower left) of the full-length sodB gene Campylobacter UPTC CF89-12 UN C. lari RM2100 C. jejuni NCTC11168 C. coli RM2228 C. upsaliensis RM3195 UPTC CF89-12 96.7 80.8 82.0 75.9 UN C. lari RM2100 92.9 81.7 83.1 77.0 C. jejuni NCTC11168 83.6 84.0 92.4 79.1 C. coli RM2228 82.7 83.1 97.7 80.0 C. upsaliensis RM3195 78.8 78.3 84.5 84.5 1352 Ann Microbiol (2014) 64:1347–1356 Table 3 PCR amplifications of the sodB and sodC genes segments in adjacent genetic loci in the UPTC CF89-12 (AB736167; C. lari. NCTC National Collection of Type Cultures; JCM Japan Col- AB736168), UN C. lari RM2100 (NC_012039) and some lection of Microorganisms; NA not available other thermophilic Campylobacter strains, C. jejuni Campylobacter lari Source Country sodB sodC NCTC11168, C. coli RM2228 and C. upsaliensis RM3195 are shown schematically in Figs. 3 and 4. UPTC NCTC12892 River water England + + UPTC NCTC12894 Sea water England + + Comparative analyses of putative sodB and sodC gene UPTC CF89-12 River water Japan + + clusters UPTC CF89-14 River water Japan + + UPTC A1 Seagull N Ireland + + As described above, putative full-length sodB and sodC UPTC 89049 Human France + + genes clusters were found in the UPTC CF89-12 strain UPTC 92251 Human France + + (Figs. 3, 4). A possible open reading frame (ORF) UPTC 2 Oyster Northen Ireland + + [nucleotide position (np) 6,900–7,550 bp; AB736167] UPTC 27 Mussel Northen Ireland + + for sodB and a possible ORF (np 1,882–2,418 bp; UPTC 136 Scallop Northen Ireland + + AB736168) for sodC were identified. In the present UPTC 150 Cockle Northen Ireland + + study, the nucleotide positions used are for those of UPTC 182 Sea water Northen Ireland + + UPTC CF89-12 (AB736167 for sodB and AB736168 UPTC 476 Mussel Northen Ireland + + for sodC). These were predicted to encode peptides of UPTC 504 Mussel Northen Ireland + + 217 and 179 amino acid residues with calculated mo- UN C. lari JCM2530 Seagull Japan + + lecular weights (CMWs) of 24.1 and 19.9 kDa, respec- UN C. lari 28 Mussel Northen Ireland + + tively. These two possible ORFs of sodB and sodC UN C. lari 34 NA Northen Ireland + + genes were identified, based on comparison of nucleo- UN C. lari 170 Seagull Japan + + tide and deduced amino acid sequence similarities with UN C. lari 175 Seagull Japan + + those of the corresponding genes from UN C. lari UN C. lari 176 Black-tail gull Japan + + RM2100 (NC_012039) (sodB and sodC)and C. jejuni NCTC11168 (NC_002163) (sodB). UN C. lari 264 Mussel Northen Ireland + + UN C. lari 274 Mussel Northen Ireland + + Molecular and comparative analyses of sodB and sodC genes UN C. lari 295 Human Canada + + in other thermophilic Campylobacter organisms UN C. lari 298 Human Canada + + UN C. lari 299 Human United States + + As shown in Figs. 3 and 4, sodB and sodC genes are located UN C. lari 300 Seagull United States + + approximately 55–56 kbp from each other within genomic UN C. lari 448 Mussel Northen Ireland + + DNA in the UN C. lari RM2100 strain. In the UPTC CF89-12 UN C. lari 84C-1 Human Northen Ireland + + strain, these two genes are also located in a similar manner (data not shown). sodB and sodC genes in UPTC CF89-12 strain shared relatively high nucleotide sequence similarity (92.9 % and gene segments with the C. lari isolates (n =14 for UPTC and 91.7 %) with those in the UN C. lari RM2100 strain, respec- n =14 for UN C. lari; Table 3), as shown in Fig. 5. tively. In addition, sodB genes in UPTC CF89-12 and UN The primer pairs used generated the expected C. lari RM2100 strains shared relatively low nucleotide se- amplicons for sodB (Fig. 5a)and sodC (Fig. 5b)gene quence similarities with those in C. jejuni NCTC11168 segments with all 28 C. lari isolates, respectively. Thus, the present results indicate strongly that both the sodB (80.8 % and 81.7 %), C. coli RM2228 (82.0 % and 83.1 %) and C. upsaliensis RM3195 (75.9 % and 77.0 %), respective- and sodC genes are conserved within the C. lari iso- ly. These are shown in Table 2. lates genomic DNA, consisting of the UPTC and UN C. lari taxa (Table 3). However, no sodC was identified PCR amplification of sodB and C gene segments within the other three C. jejuni , C. coli and C. upsaliensis reference strains (data not shown). Thus, Two primer pairs [f-/r-ClsodB and f-/r-ClsodC (Figs. 1 and 2)] C. lari organisms carry sodB and sodC gene loci at were employed for PCR amplification of the sodB and sodC extremely high frequency. Ann Microbiol (2014) 64:1347–1356 1353 M 1321 4 5 6 7 8 90111213 14 M15 16 17 18 19 20 21 22 23 24 25 26 27 28 bp bMM 1321 4 5 6 7 8 90111213 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 bp Fig. 5 Agarose gel electrophoresis profiles of PCR amplicons of a sodB 100 bp DNA ladder (New England BioLabs), 1–28, refer to order in and b sodC genes segments amplified using two sets of primer pairs, f-/r- Table 3 (UPTC NCTC12892–UN C. lari 84C-1) ClsodB and f-/r-ClsodC, respectively, with C. lari isolates. Lanes: M SOD activity determination Discussion Next, SOD activities were determined in C. lari isolates, In the present study, sodB and sodC gene clusters were iden- including UPTC, and compared with those of other thermo- tified in UPTC CF89-12. The genetic loci of sodB gene clusters philic Campylobacter organisms. In Table 4,the SOD activ- and their adjacent genetic loci in UPTC CF89-12 (AB736167; ities are shown with 20 C. lari isolates (n=10 for UPTC; n= AB736168), UN C. lari RM2100 (NC_012039) and some 10 for UN C. lari isolates). other thermophilic Campylobacter strains, C. jejuni The SOD activities were also measured with the other three NCTC11168, C. coli RM2228 and C. upsaliensis RM3195, thermophilic Campylobacter organisms of C. coli (n =10), were completely different (Figs. 3, 4). Relatively high sequence C. jejuni (n =10) and C. upsaliensis (n =9) (Table 4). As shown similarities of sodB geneswereseeninthese Campylobacter in Table 4, almost all the 28 isolates out of a total of the 29 organisms, both at the nucleotide and amino acid sequence demonstrated low-SOD activity (<0.15 U/μg). Thus, the 13 levels (Table 3). In addition, C. jejuni NCTC11168, C. coli C. lari isolates (n =4 for UPTC; n =9 for UN C. lari isolates) RM2228 and C. upsaliensis RM3195 did not carry the sodC out of a total of the 20 C. lari isolates showed high-SOD gene. Although bacterial SOD may support the ability to chal- activity (Table 4). In addition, in the present study, more high- lenge the host immune system, and therefore may be consid- SOD activity was seen with the nine C. lari isolates including ered as a virulence determinant, this result suggests that not UPTC than was seen with the other three thermophilic having a sodC gene is not associated with virulence in Campylobacter and Helicobacter pylori organisms (Table 4). Campylobacter organisms. 1354 Ann Microbiol (2014) 64:1347–1356 Table 4 Superoxide dismutase (SOD) activity determination of C. lari Table 4 (continued) and other bacterial isolates. C, Campylobacter; H, Helicobacter; H,high Isolate SOD activity Remarks activity; M,moderate activity; L, low activity (U/μgprotein) Isolate SOD activity Remarks (U/μgprotein) C. upsaliensis 29-3 0.0171±0.0008 L C. upsaliensis 13-1 0.0711±0.0022 L C. lari UPTC NCTC12892 0.1394±0.0422 L C. upsaliensis 21-1 0.1233±0.0038 L C. lari UPTC NCTC12894 0.1401±0.0051 L C. upsaliensis faline 104-1 0.0817±0.0057 L C. lari UPTC CF89-12 0.3931±0.0139 H C. upsaliensis faline 37-1 0.1396±0.0250 L C. lari UPTC A1 0.0684±0.0046 L C. upsaliensis LMG8850 0.0714±0.0049 L C. lari UPTC 89049 0.3226±0.0520 H C. upsaliensis G1 0.2226±0.0731 H C. lari UPTC 92251 0.2921±0.0239 H H. pylori KMT52 0.326±0.021 H C. lari UPTC 27 0.2221±0.0229 H H. pylori KMT97 0.199±0.003 M C. lari UPTC 88 0.0694±0.0097 L H. pylori NY14 0.031±0.006 L C. lari UPTC 136 0.1599±0.0236 M C. lari UPTC 150 0.1759±0.0009 M Morishita et al. (2012) C. lari JCM2530 0.1216±0.0292 L C. lari 28 0.4637±0.0084 H C. lari 170 0.4560±0.0118 H Probable ribosome-binding (RB) sites (Shine-Dalgarno se- C. lari 176 0.3917±0.0076 H quences; Benjamin 2000) that are complementary to a highly C. lari 264 0.3677±0.0405 H conserved sequence of CCUCCU close to the 3′-end of 16S C. lari 274 0.3463±0.0224 H rRNA, AGGAGA (np 6,889–6,894 bp) for sodB and AGGA C. lari 295 0.4492±0.0111 H GA (np 1,871–1,876 bp) for sodC were identified in UPTC C. lari 298 0.2737±0.0121 H CF89-12 (Fig. 6). Two putative promoters, consisting of con- C. lari 299 0.3931±0.0055 H sensus sequences at the −35 (TTGAAA; np 6,823–6,828 bp) C. lari 84C-1 0.4638±0.0006 H and −10 (TATAAA; np 6,875–6,880 bp)-like structures, were C. coli 13 0.0136±0.0010 L also identified immediately upstream of the sodB gene in C. coli 24 (DO24) 0.0592±0.0000 L UPTC CF89-12 (Fig. 6). −35 (TTGACC; np 1,780– C. coli 27 0.0557±0.0047 L 1,785 bp) and −10 (TATTAT; np 1,838–1,843 bp)-like pro- C. coli 48 (PG48) 0.0815±0.0082 L moters also occurred upstream of sodC. Regarding the sodC C. coli 110 0.0634±0.0119 L gene in UPTC CF89-12, a putative intrinsic ρ-independent C. coli 154 0.0424±0.0018 L transcriptional terminator occurred immediately downstream C. coli JCM2529 0.0574±0.0014 L of the stop codon for the sodC gene. C. coli PG20 0.0325±0.0026 L The putative anaerobic ribonucleoside triphosphate C. coli 165 0.0691±0.0023 L reductase activating protein (No. 3 in Fig. 3a,c)gene C. coli 23 (DO23) 0.0752±0.0008 L (np 7,456–8,508 bp), immediately downstream of the C. jejuni 81-176 0.0753±0.0020 L sodB gene, was identified with the possible ORF (np C. jejuni LCDC4483 0.1041±0.0011 L 7,456–8,505 bp) in UPTC CF89-12. The probable RB C. jejuni HP5090 0.0391±0.0044 L site AGGAAA (np 7,446–7,451 bp) for the putative C. jejuni HP5095 0.0592±0.0001 L protein gene was also present. The putative anaerobic ribonucleoside triphosphate reductase gene (No. 2 gene C. jejuni D3083 0.0652±0.0028 L C. jejuni ST23 0.0845±0.0065 L in Fig. 3a) (np 8,510–9,025 bp) also occurred down- stream of theputativegene(No.3gene in Fig. 3a). In C. jejuni LMG6444 0.0478±0.0017 L addition, the putative conserved hypothetical protein C. jejuni HP5084 0.0573±0.0152 L (DUF 285 domain protein) gene (No. 1 gene in C. jejuni 81116 0.0799±0.0046 L Fig. 3a) (np 9,022–9,417 bp) also occurred downstream C. jejuni 79AH88-88 0.0733±0.0000 L of the putative gene. Two RB sites, AGGA (np 8,500– C. upsaliensis Maliryn 0.0108±0.0046 L 8,503 bp) and AAGAG (np 9,006–9,010 bp) were also C. upsaliensis 41-2 0.0411±0.0042 L identified for these hypothetical and putative genes. Ann Microbiol (2014) 64:1347–1356 1355 T-rich region -10 like region RBS Start codon -AAAATAAAGACTCATTTAGTCTTTTTTAATTTAACTATGTTAGAATGCTATAAAATTTAAAAAAGGAGAACAAAATGTTT C. lari RM2100 50,266 50,345 A..........G.TG.................................T..........-..............T...... UCPT CF89-12 3,701 3,780 ********** * ********************************* ********** ************** ****** T-rich region -10 like region RBS Start codon C. lari RM2100 106,350 TTTTATAAGTTTTTTGATTATTATATTTTATAAAAAATAATTTTTCATTAATAATATATGTTACAATAAAATAA-TTTTAACAAAAGGAGAAAAAATGAAA 103,251 -............................................T.......T....................T................G......... UCPT CF89-12 1,788 1,887 ******************************************** ******* ******************** **************** ********* c d TT TT GT GC T T AT TA TA CG TA GC CG ACT AAAGCT TT AGT AT TA 2,436 2,447 AA AAATCGTTTTACTC 4,438 4,455 Fig. 6 Nucleotide sequence alignment analyses of the putative promoter genes from UPTC CF89-12 and C. lari RM2100. Nucleotide sequences structures, consisting of T-rich and −10 regions, as well as the ribosome were also examined for the transcriptional terminator structures for c binding site (RBS) and the start codon ATG, for a sodB and b sodC sodB and d sodC. For the others, refer to the legend to Fig. 3 Previously, Kikuchi and Suzuki (1984) found very Overall, C. lari organisms carry both sodB and sodC genes high SOD activities in Campylobacter strains, especially within their genome and may have higher SOD activities than in C. fetus subsp. fetus compared with those in the other three thermophilic Campylobacter and H. pylori Escherichia coli , Propionibacterium acnes and organisms (Morishita et al. 2012), which carry only one Veillonella alcalescens and Pesci et al. (1994)suggested sodB gene but no sodC. Therefore, C. lari may have an a potential role for sodB in C. jejuni intracellular sur- advantage over other thermophilic Campylobacter organisms vival. In addition, SOD is an important determinant in for survival strategies within their host environment. the ability of C. coli to survive aerobically and for optimal colonization within the chicken gut (Purdy Acknowledgments This research was supported by partially a project grant funded by a Grant-in-Aid for Scientific Research (C) (20580346) et al. 1999); however, no SOD activity studies on C. from the Ministry of Education, Culture, Sports, Science and Technology lari organisms, specific for genetically variable and di- of Japan (to M.M.). M.M. and J.E.M. were funded through a Great Britain verse species (Miller et al. 2008), have appeared in the Sasakawa Foundation (Butterfield) Award to jointly examine the clinical literature. significance of Campylobacter infection in the UK and Japan. Recently, Morishita et al. (2012) showed that SOD activi- ties of all 158 Helicobacter pylori isolates [n =2 for se- quenced strains 26695 and J99; n =156 from clinical isolates References from 156 Japanese patients with gastroduodenal diseases such as gastric cancer (n =59) and non-cancer (n =97)] by SOD Atack JM, Kelly DJ (2009) Oxidative stress in Campylobacter jejuni: Assay Kit-WST (Dojindo Molecular Technologies) were di- responses, resistance and regulation. Future Microbiol 4:677–690 vided into three groups: high SOD activity (>0.22 U/μg n =2; Atack JM, Harvey P, Jones MA, Kelly DJ (2008) The Campylobacter only 3 % occurrence), moderate SOD activity (0.15≦≦0.22, jejuni thiol peroxidases Tpx and Bcp both contribute to aerotolerance and peroxide-mediated stress resistance but have dis- n =16) and low SOD activity (<0.15, n =140). However, in tinct substrate specificities. J Bacteriol 190:5279–5290 the present study, 13 C. lari isolates out of a total of the 20 C. Benjamin L (2000) Genes VII. Oxford University Press, Oxford lari (n =10 UN C. lari; n =10 UPTC isolates) showed high Benjamin J, Leaper S, Owen RJ, Skirrow MB (1983) Description of SOD activity (Table 4). Campylobacter laridis, a new species comprising the nalidixic acid 1356 Ann Microbiol (2014) 64:1347–1356 resistant thermophilic Campylobacter (NARTC) group. Curr laridisvariant) isolated from an appendix and from human feces. J Microbiol 8:231–238 Clin Microbiol 26:1050–1051 Bezian MC, Ribou G, Barberis-Giletti C, Megraud F (1990) Isolation of a Miller WG, Wang G, Binnewies TT, Parker CT (2008) The complete urease positive thermophilic variant of Campylobacter lari from a genome sequence and analysis of the human pathogen patient with urinary tract infection. Eur J Clin Microbiol Infect Dis 9: Campylobacter lari. Foodborne Pathog Dis 5:371–386 895–897 Morishita K, Takeuchi H, Morimoto N, Shimamura T, Kadota Y, Tsuda M, Blaser MJ, Taylor DN, Feldman RA (1983) Epidemiology of Taniguchi T, Ukeda H, Yamamoto T, Sugiura T (2012) Superoxide Campylobacter jejuni infections. Epidemiol Rev 5:157–176 dismutase activity of Helicobacter pylori perse from 158 clinical Bolton FJ, Holt A, Hutchinson DN (1985) Urease-positive thermophilic isolates and the characteristics. Microbiol Immunol 56:262–272 campylobacters. Lancet 1:1217–1218 Nachamkin I, Stowell C, Skalina D, Jones AM, Hoop RM 2nd, Smibert Endtz HP, Vliegenthart JS, Vandamme P, Weverink HW, van den Braak RM (1984) Campylobacter laridis causing bacteremia in an immu- NP, Verbrugh HA, van Belkum A (1997) Genotypic diversity of nosuppressed patient. Ann Int Med 101:55–57 Campylobacter lari isolated from mussels and oysters in The Owen RJ, Costas M, Sloss L, Bolton FJ (1988) Numerical analysis of Netherlands. Int J Food Microbiol 34:79–88 electrophoretic protein patterns of Campylobacter laridis and allied Harrington CS, Thomson-Carter FM, Carter PE (1997) Evidence for thermophilic campylobacters from the natural environment. J Appl recombination in the flagellin locus of Campylobacter jejuni:im- Bacteriol 65:69–78 plications for the flagellin gene typing scheme. J Clin Microbiol 35: Palyada K, Sun YQ, Flint A, Butcher J, Naikare H, Stintzi A (2009) 2386–2392 Characterization of the oxidative stress stimulon and PerR regulon Hwang S, Kim M, Ryu S, Jeon B (2011) Regulation of oxidative stress of Campylobacter jejuni. BMC Genomics 10:481 response by CosR, an essential response regulation in Pesci EC, Cottle DL, Pickett CL (1994) Genetic, enzymatic, and patho- Campylobacter jejuni. PLoS One 6:e22300 genic studies of the iron superoxide dismutase of Campylobacter Kaneko A, Matsuda M, Miyajima M, Moore JE, Murphy PG (1999) jejuni. Infect Immun 62:2687–2694 Urease-positive thermophilic strains of Campylobacter isolated Purdy D, Cawthraw S, Dickinson JH, Newell DG, Park SF (1999) from seagulls (Larus spp.). Lett Appl Microbiol 29:7–9 Generation of a superoxide dismutase (SOD)-deficient mutant of Kikuchi H, Suzuki T (1984) An electrophoretic analysis of superoxide Campylobacter coli: evidence for the significance of SOD in cam- dismutase in Campylobacter spp. J Gen Microbiol 130:2791–2796 pylobacter survival and colonization. Appl Environ Microbiol 65: Martinot M, Jaulhac B, Moog R, De Martino S, Kehrli P, Monteil H, 2540–2646 Piemont Y (2001)Campylobacter lari bacteremia. Clin Microbiol Scott MD, Meshnick SR, Eaton JW (1987) Super oxide dismutase-rich Infect 7:96–97 bacteria. J Biol Chem 262:3640–3645 Matsuda M, Moore JE (2004) Urease-positive thermophilic Skirrow MB, Benjamin J (1980) ‘1001’ campylobacters: cultural charac- Campylobacter species. Appl Environ Microbiol 70:4415–4418 teristics of intestinal campylobacters from man and animals. J Hyg Matsuda M, Kaneko A, Fukuyama M, Itoh T, Shingaki M, Inoue M, (Camb) 85:427–442 Moore JE, Murphy PG, Ishida Y (1996) First finding of urease- Storz G, Imaly JA (1999) Oxidative stress. Curr Opin Microbiol 2:188– positive thermophilic strains of Campylobacter in river water in the 194 Far East, namely, in Japan, and their phenotypic and genotypic Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving characterization. J Appl Bacteriol 81:608–612 the sensitivity of progressive multiple sequence alignment through Matsuda M, Shibuya T, Itoh Y, Takiguchi M, Furuhata K, Moore JE, sequence weighting, position-specific gap penalties and weight ma- Murayama O, Fukuyama M (2002) First isolation of urease-positive trix choice. Nucleic Acids Res 22:4673–4680 thermophilic Campylobacter (UPTC) from crows (Corvus van Vliet AHM, Ketley JM, Park SF, Penn CW (2002) The role of iron in levaillantii) in Japan. Int J Hyg Environ Health 205:321–324 Campylobacter gene regulation, metabolism and oxidative stress Matsuda M, Kaneko A, Stanley T, Millar BC, Miyajima M, Murphy PG, defense. FEMS Microbiol Rev 26:173–186 Moore JE (2003) Characterization of urease-positive thermophilic Werno AM, Klena JD, Shaw GM, Murdoch DR (2002) Fatal case of Campylobacter subspecies by multilocus enzyme electrophoresis Campylobacter lari prosthetic joint infection and bacteremia in an typing. Appl Environ Microbiol 69:3308–3010 immunocompetent patient. J Clin Microbiol 40:1053–1055 Mégraud F, Chevrier D, Desplaces N, Sedallian A, Guesdon JL (1988) Wilson IG, Moore JE (1996) Presence of Salmonella spp. and Urease-positive thermophilic campylobacter (Campylobacter Campylobacter spp. in shellfish. Epidemiol Infect 116:147–153 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Molecular analysis of superoxide dismutase in Campylobacter lari

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Springer Journals
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Copyright © 2013 by Springer-Verlag Berlin Heidelberg and the University of Milan
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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1869-2044
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10.1007/s13213-013-0778-7
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Abstract

Ann Microbiol (2014) 64:1347–1356 DOI 10.1007/s13213-013-0778-7 ORIGINAL ARTICLE Molecular analysis of superoxide dismutase in Campylobacter lari Takuya Nakajima & Takashi Kuribayashi & Shizuo Yamamoto & John E. Moore & Beverley C. Millar & Motoo Matsuda Received: 29 July 2013 /Accepted: 18 November 2013 /Published online: 12 December 2013 Springer-Verlag Berlin Heidelberg and the University of Milan 2013 Abstract The superoxide dismutase (SOD) gene clusters, segments with 28 C. lari isolates, including 14 UPTC isolates, sodB and sodC, and their adjacent genetic loci from a urease- gave positive results. C. lari organisms were shown to carry positive thermophilic Campylobacter (UPTC) CF89-12 strain both the sodB and sodC genes with extremely high frequency. were analyzed molecularly, and compared with those of ther- More high-SOD activity was seen with the C. lari isolates (n = mophilic campylobacters. The UPTC CF89-12 strain carried 9), including UPTC, than was seen with the other three ther- sodB [structural gene 654 base pairs (bp)] and sodC (540 bp) mophilic Campylobacter and Helicobacter pylori organisms. genes, as did the Campylobacter lari RM2100 reference strain. . . However, the other three thermophilic Campylobacter jejuni, Keywords Oxidative stress defense SOD Thermophilic . . C. coli and C. upsaliensis reference strains carried only a single campylobacters C. lari Urease-positive thermophilic sodB gene, and no sodC. Although sodB and sodC in the Campylobacter UPTC strain shared relatively high nucleotide sequence simi- larities (92.9%and91.7%, respectively) with the correspond- ing genes in the C. lari RM2100 strain, the sodB gene in the Introduction UPTC CF89-12 and C. lari RM2100 strains shared relatively low nucleotide sequence similarities with those in C. jejuni Oxidative stress is an important matter for organisms NCTC11168 (80.8 % and 81.7 %), C. coli RM2228 (82.0 % employing oxygen as a terminal electron acceptor, since the and83.1%)and C. upsaliensis RM3195 (75.9 % and 77.0 %), combination of oxygen and an electron can generate reactive respectively. All PCR amplifications of sodB and sodC gene oxygen such as the superoxide (O ), the hydroxyl radical (HO⋅) and hydrogen peroxide (H O ) (Storz and Imaly 1999). 2 2 These reactive oxygen species can lead to damage of proteins, T. Nakajima M. Matsuda (*) nucleic acids and membranes (Atack et al. 2008). Reactive Laboratory of Molecular Biology, Graduate School of Environmental oxygen species are also generated by the immune system to Health Sciences, Azabu University, Sagamihara 252-5201, Japan kill invading microbes (Atack et al. 2008). Therefore, for e-mail: matsuda@azabu-u.ac.jp bacterial pathogens to survive, they must resist the reactive T. Kuribayashi S. Yamamoto oxygen stress encountered in both the host and the environ- Laboratory of Immunology, Graduate School of Environmental ment. Recent studies suggest that bacteria contain a wide Health Sciences, Azabu University, Sagamihara 252-5201, Japan range of enzymes involved in oxidative stress (Atack and J. E. Moore B. C. Millar Kelly 2009). Superoxide dismutase (SOD) is considered im- Department of Bacteriology, Northern Ireland Public Health portant in protection of aerobes against oxidant damage (Scott Laboratory, Belfast City Hospital, Belfast BT9 7 AD, UK et al. 1987). Thermophilic Campylobacter jejuni and Campylobacter J. E. Moore School of Biomedical Sciences, University of Ulster, coli species are curved, Gram-negative organisms, and are Londonderry BT52 1SA, Northern Ireland, UK the most commonly recognized causes of acute bacterial diarrhea in the Western world (Skirrow and Benjamin 1980; J. E. Moore Benjamin et al. 1983; Blaser et al. 1983). Thermophilic Centre for Infection and Immunity, Queen’s University, Belfast BT9 7AB, Northern Ireland, UK Campylobacter lari species was first isolated particularly 1348 Ann Microbiol (2014) 64:1347–1356 Table 1 Thermophilic Campylobacter isolates analyzed in the present from seagulls (Skirrow and Benjamin 1980; Benjamin et al study and accession numbers of nucleotide sequence data of sod genes 1983). C. lari has also been shown to occasionally be a cause clusters accessible in the DDBJ/EMBL/GenBank of clinical infection (Nachamkin et al. 1984;Martinot etal. Campylobacter Source Country Accession no. 2001; Werno et al. 2002). In addition, an atypical group of urease-positive thermophilic Campylobacter (UPTC) organ- UPTC CF89-12 River water Japan AB736167 isms have been isolated from the natural environment in C. lari RM2100 Human United States NC_012039 England in 1985 (Bolton et al. 1985). Thereafter, these organ- C. jejuni NCTC11168 Human England NC_002163 isms were described as a biovar or variant of C. lari (Bolton C. coli RM2228 Chicken United States AAFL01000005 et al. 1985; Mégraud et al. 1988). Subsequent isolates were C. upsaliensis RM3195 Human United States AAFJ01000003 reported in Europe (Mégraud et al. 1988;Owen etal. 1988; Bezian et al. 1990; Wilson and Moore 1996;Kaneko etal. 1999; Endtz et al. 1997; Matsuda et al. 2003); and in Japan (Matsuda et al. 1996; Matsuda et al. 2002). Thus, at least these thermophilic Campylobacter reference strains analyzed in two representative taxa, namely urease-negative (UN) C. lari the present study and the accession numbers of the nucleotide and UPTC exist within the species C. lari (Matsuda and sequence data of their sodB and sodC genes clusters are also Moore 2004). shown in Table 1. Regarding the oxidative stress defense system in Campylobacter cells were cultured on Mueller-Hinton Campylobacter, van Vlietetal. (2002) reviewed the role of agar (Oxoid, Hampshire, UK) containing defibrinated iron in Campylobacter gene regulation, metabolism and oxi- horse blood [7 % (v/v); Nippon Bio-test, Tokyo, Japan], supplemented with Butzler Campylobacter-selec- dative stress defense. In addition, Atack et al. (2008)sug- gested that thioredoxin-linked thiol peroxidase and tive medium (Virion, Zurich, Switzerland), under bacterioferritin comigratory protein are partially redundant microaerophilic conditions produced by BBL Campypak antioxidant enzymes that play an important role in protection Microaerophilic System Envelopes (Becton Dickinson, of C. jejuni against oxygen-induced oxidative stress. More Franklin Lakes, NJ) at 37 °C for 48 h. Cells were further recently, oxidative stress in C. jejuni, responses, resistance cultured on Mueller-Hinton agar under the same and regulation (Atack and Kelly 2009), characterization of the microaerophilic conditions. oxidative stress stimulon and peroxide-sensing regulator regulon of C. jejuni (Palyada et al. 2009) and regulation of the oxidative stress response by the Campylobacter oxidative Genomic DNA preparation stress regulator—an essential response regulator in C. jejuni Template DNA was prepared using sodium dodecyl sulfate (Hwang et al. 2011)—were reported. Regarding the oxidative stress defense system in C. lari, and proteinase K treatment, phenol-chloroform extraction and Miller et al. (2008) described recently sodB and sodC in UN ethanol precipitation (Harrington et al. 1997), and adjusted to C. lari RM2100 strain following analysis of the complete approximately 500 ng/μL. genome sequence in this organism. However, to our knowl- edge, the system has not yet been described in UPTC organ- isms. Therefore, the present study aimed to identify sod genes Construction of the genomic DNA library of the UPTC clusters in the Campylobacter lari organisms, including CF89-12 strain and nucleotide sequence determination UPTC isolates, and to perform genotypic and phenotypic TM comparisons of the SOD genes within thermophilic A genomic DNA library was constructed using NEBNext campylobacters. DNA Sample Prep. Reagent Set 1 (New England BioLabs, Japan, Tokyo, Japan). The DNA was fragmented using Covaris S-Series (Covaris, Woburn, MA) and separated by Materials and methods agarose gel electrophoresis [300–500 base pairs (bp)]. Cluster generation was carried out using the constructed library DNA Campylobacter lari isolates and growth conditions used as templates with Cluster Station and Cluster Generation Kit in the present study (Illumina, San Diego, CA). The nucleotide sequence (sequence reads 75 bp) was de- The Japanese strain, UPTC CF89-12 (Table 1), which was termined using Genome Analyzer IIx and Sequencing Kit isolated from the water of the Asahigawa River, Okayama (Illumina). De novo assembly of the paired-end reads prefecture, Japan (Matsuda et al. 1996), was employed for the (35 bp) was carried out using Edena (V2.1.1., http://www. construction of a genomic DNA library, cloning, sequencing genomic.ch/edena.php) and Velvet (V0.7.11, http://www.ebi. ac.uk/~zerbino/velvet/). and characterization of the sod genes clusters. Other Ann Microbiol (2014) 64:1347–1356 1349 f-ClsodB ATGTTTGAATTAAGAAAACTACCTTATGAAGCAGATGCTTTTGGAGACTTTTTAAGTGCAGAAACTTTTGCGTTTCATCATGGCAAACACCACCAAACTTATGTAAATAAC 50,450 C. lari RM2100 50,340 ..................T................C...........T........C...........C..A....................................... 3,885 UPTC CF89-12 3,375 C. jejuni NCTC11168 166,373 ..................T..........TA.CA..........T..T.....G.....T.........AGC.A.........A.....T...A.T........T.CA..T 166,483 C. coli RM2228 49,969 ..................T..........TA.CA..........T..T...........T.........AGT.A.........A.........A.T..........CA..T 50,079 110,491 C. upsaliensis RM3195 110,381 ..................T.......C..TA.TA..........C..T...........T..G..C...AGT.A......C.......................T.....T ****************** ******* ** * * ******** ** ***** ** ** ** ** ** * ****** ** ***** *** * ******** * ** r-ClsodB 50,993 C. lari RM2100 50,886 TACGCGCACATTAACTGGGAATTCGTAGCAAAAGCTTATGAATGGGCTATCAAAGAAGGTATGAACTCAGTAAGTTTTTATGCAAACGAATTGCACCCTGTAAAATAA .....A..T..............T..................................................C...............C.T..T..AC....C... 4,428 UPTC CF89-12 4,321 ..T..T..T..............T..T...........C.........T.A........C...GGA.....T..C...........T...C.T............... 167,035 C. jejuni NCTC11168 166,918 C. coli RM2228 50,524 ..T..T..T..............T........................T.A............GGA.....T..C.....C.........C.T..T............ 50,631 111,022 C. upsaliensis RM3195 110,915 .....T..T..........CT..T..T..............G......T.G.....G.......G......T..C.....C.....T...C.A........G...... ** ** ** ********** ** ** *********** ** ****** * ***** ** *** ***** ** ***** ***** *** * ** ** * ** *** f-ClsodC UNC. lari RM2100 106,256 ATGAAAAAAATAATTATAGGCTCTTTACTAGCATCAAGCTTTTTAATTGGGGCAAATTTAGAAAATTTTGATCCAAAAGCACAAAAAGATCATTTAGTTA 106,157 ...........T..AC.............G........T..C.......................................................... 1,981 UPTC CF89-12 1,882 *********** ** ************* ******** ** ********************************************************** r-ClsodC 105,717 UNC. lari RM2100 105,756 CGGTGGTGGCGCTAGAATGGCTTGTGGAGTTATTAAGTAA .........T.................G.A.TA.TCC... 2,421 UPTC CF89-12 2,382 ********* ***************** * * * *** Fig. 1 a Nucleotide sequence alignment analyses of the putative sodB of the sodB gene was omitted from the strains, respectively (nt 3,886– gene in the UN Campylobacter lari RM2100, C. lari UPTC CF89-12, C. 4,320 bp for UPTC CF89-12). b Nucleotide sequence alignment analyses jejuni NCTC11168, C. coli RM2228 and C. upsaliensis RM3195 strains. of the sodC gene in the UN C. lari RM2100 and UPTC CF89-12 strains. Numbers at the left and right refer to the nucleotide positions determined. A central region of approximately 400 bp (nt 1,982–2,381 bp for the Dots Identical bases (changes are explicit), dashes deletions; asterisks UPTC CF89-12) of the sodC gene was omitted identical positions in all cases. A central region of approximately 430 bp Putative sodB and sodC gene sequence analyses comparison of nucleotide and deduced amino acid sequence similarities with those of the corresponding Sequence analyses of the putative sodB and sodC genes and sodB and sodC genes from the UN C. lari RM2100 their adjacent genetic loci from the UPTC CF89-12 strain strain (DDBJ/ EMBL/GenBank accession no. were carried out using the GENETYX-Windows computer NC_012039). software (version 9; GENETYX, Tokyo, Japan). Nucleotide sequence alignment analyses of the puta- The putative sodB and sodC genes sequences from tive sodB and sodC from the UPTC CF89-12 strain the UPTC CF89-12 strain were identified based on were carried out with the accessible sequence data of Fig. 2 Schematic representations of the putative sodB (a)and sodC (b) genes and their adjacent genetic loci in the UPTC CF89-12 strain, showing the locations of 12 3 4 6 the primer pairs of f-/r-ClsodB f-ClsodB r-ClsodB and f-/r-ClsodC designed in silico for the sodB and sodC genes segments, and their sequences (c). The gene numbers (1–6) in the box correspond to those in Figs. 3a,c and 4a,c 12 3 4 5 f-ClsodC r-ClsodC Primer Nucleotide sequence (5'-3') CAAACACCACCAAACTTATG f-ClsodB r-ClsodB GCCCATTCATAAGCTTTTGC GGCTCTTTACTGGCATCAAG f-ClsodC ACCCCACAAGCCATTCTAGC r-ClsodC 1350 Ann Microbiol (2014) 64:1347–1356 a C. lari UPTC CF89-12 (AB736167) 12 3 4 6 b UN C. lari RM2100 (NC_012039) 12 3 4 5 6 UPTC CF89-12 C. lari RM2100 No. Gene Product 5' end 3' end 5' end 3' end 1 201 815 46,766 47,380 Cla_0047 conserved hypothetical protein (DUF285 domain protein) 2 948 3,227 47,512 49,791 nrdD anaerobic ribonucleoside triphosphate reductase 3 3,175 3,708 49,739 50,272 Cla_0049 anaerobic ribonucleoside triphosphate reductase activating protein 4 3,775 4,428 50,340 50,993 sodB superoxide dismutase (Fe) 5 - - 51,053 52,426 Cla_0051 putative C4-dicarboxylate transporter 6 5,448 4,459 53,438 52,449 purM phosphoribosylaminoimidazole synthetase d C. upsaliensis RM3195 (AAFJ01000003) 1326 4 5 C. upsaliensis RM3195 No. Gene Product 5' end 3' end 1 108,266 106,944 clpP ATP-dependent Clp protease, proteolytic subunit ClpP 2 108,371 108,955 folE GTP cyclohydrolase I 3 108,966 110,093 CUP1761 hypothetical protein 4 110,381 111,022 CUP1762 superoxide dismutase (sodB) 5 111,646 111,023 CUP1763 phosphoribosyl-ATP pyrophosphatase/phosphoribosyl-AMP cyclohydrolase 6 111,951 111,643 CUP1764 TfoX domain protein, putative f C. jejuni NCTC11168 (NC_002163) 12 5 6 7 8 c C. coli RM2228 (AAFL01000005) 15 34 7 8 C. jejuni NCTC11168 C. coli RM2228 No. Gene Product 5' end 3' end 5' end 3' end 1 165,580 165,017 48,624 48,061 NA putative integral membrane protein 2 166,105 165,938 - - NA putative periplasmic protein 3 - - 49,032 49,439 NA integral membrane protein, putative 4 - - 49,570 49,863 NA integral membrane protein, putative 5 166,373 167,035 49,969 50,631 sodB superoxide dismutase (fe) 6 167,050 167,794 - - NA hypothetical protein 7 167,807 169,012 51,863 50,658 NA putative saccharopine dehydrogenase 8 169,962 169,054 52,804 51,899 NA putative iron-uptake ABC transporter ATP-binding protein Fig. 3 Schematic representations of the putative sodB gene and its coli RM2228. Further details shown in c, e and h are 5′ end (np, bp) and adjacent genetic loci in strains a C. lari UPTC CF89-12, b UN C. lari 3′ end (np, bp); − absent, NA not available RM2100, d C. upsaliensis RM3195, f C. jejuni NCTC11168 and g C. other thermophilic Campylobacter strains, UN C. lari upsaliensis RM3195 (AAFJ01000003) using CLUSTAL W RM2100 (NC_012039), C. jejuni NCTC11168 software (1.7 program) (Thompson et al 1994), which is (NC_002163), C. coli RM2228 (AAFL01000005) and C. incorporated in the DDBJ. Ann Microbiol (2014) 64:1347–1356 1351 Fig. 4 Schematic C. lari UPTC CF89-12(AB736168) representations of the putative sodC gene and its adjacent genetic loci in a C. lari UPTC CF89-12 and b UN C. 12 3 4 5 lari RM2100strains. c Details shown are 5′ end (np, bp) and 3′ end (np, bp) UN C. lari RM2100 (NC_012039) 12 3 4 5 UPTC CF89-12 C. lari RM2100 No. Gene Product 5' end 3' end 5' end 3' end 1 837 1 108,139 107,303 Cla_0124 conserved hypothetical protein, probable ATP/GTP-binding protein 2 1,778 903 107,237 106,359 Cla_0123 conserved hypothetical protein, putative transcriptional regulator (AraC family) 3 1,882 2,421 105,717 106,256 sodC superoxide dismutase (Cu/Zn) 4 2,976 2,455 105,685 105,164 Cla_0121 hypothetical protein Cla_0121 5 3,609 3,019 105,120 104,527 Cla_0120 conserved hypothetical lipoprotein Primer design for sodB and sodC gene segments ufacturer’s instructions and the procedures described by Morishita et al. (2012). One unit was defined as the Two primer pairs (f-/r-ClsodB and f-/r-ClsodC) were designed SOD amountin20 μL of a sample exhibiting 50 % inhibition in silico for amplification of the sodB and sodC genes seg- of water-soluble tetrazolium salt (WST) reduction. SOD ac- ments based on sequence information for the UPTC CF89-12 tivities were divided into three groups of high, moderate and strain (AB736167 and AB736168) and the four other thermo- low activity, according to the description by Morishita et al. philic Campylobacter reference strains shown above, in order (2012). SOD activity was determined three times in indepen- to identify the sodB and sodC genes segments in the individ- dent experiments. ual C. lari isolates (n =14 for UPTC; n =14 for UN C. lari) (Figs. 1, 2). Nucleotide sequence alignment analyses to design the Results primer pairs was carried out using CLUSTAL W soft- ware (1.7 program) (Thompson et al. 1994), as de- Molecular identification of putative sodB and sodC genes scribed above. clusters and their adjacent genetic loci in the UPTC CF89-12 strain genomic DNA Superoxide dismutase activity determination During the process of genome sequence analysis of a repre- Superoxide dismutase (SOD) activity was determined sentative taxon of C. lari UPTC, sodB and sodC genes were using the SOD-Assay Kit-WST (Dojindo Molecular identified in the genomic DNA of environmental Japanese Technologies, Kumamoto, Japan) according to the man- UPTC CF89-12. sodB and sodC genes clusters and their Table 2 Sequence similarities (%) of the nucleotide (upper right) and amino acid (lower left) of the full-length sodB gene Campylobacter UPTC CF89-12 UN C. lari RM2100 C. jejuni NCTC11168 C. coli RM2228 C. upsaliensis RM3195 UPTC CF89-12 96.7 80.8 82.0 75.9 UN C. lari RM2100 92.9 81.7 83.1 77.0 C. jejuni NCTC11168 83.6 84.0 92.4 79.1 C. coli RM2228 82.7 83.1 97.7 80.0 C. upsaliensis RM3195 78.8 78.3 84.5 84.5 1352 Ann Microbiol (2014) 64:1347–1356 Table 3 PCR amplifications of the sodB and sodC genes segments in adjacent genetic loci in the UPTC CF89-12 (AB736167; C. lari. NCTC National Collection of Type Cultures; JCM Japan Col- AB736168), UN C. lari RM2100 (NC_012039) and some lection of Microorganisms; NA not available other thermophilic Campylobacter strains, C. jejuni Campylobacter lari Source Country sodB sodC NCTC11168, C. coli RM2228 and C. upsaliensis RM3195 are shown schematically in Figs. 3 and 4. UPTC NCTC12892 River water England + + UPTC NCTC12894 Sea water England + + Comparative analyses of putative sodB and sodC gene UPTC CF89-12 River water Japan + + clusters UPTC CF89-14 River water Japan + + UPTC A1 Seagull N Ireland + + As described above, putative full-length sodB and sodC UPTC 89049 Human France + + genes clusters were found in the UPTC CF89-12 strain UPTC 92251 Human France + + (Figs. 3, 4). A possible open reading frame (ORF) UPTC 2 Oyster Northen Ireland + + [nucleotide position (np) 6,900–7,550 bp; AB736167] UPTC 27 Mussel Northen Ireland + + for sodB and a possible ORF (np 1,882–2,418 bp; UPTC 136 Scallop Northen Ireland + + AB736168) for sodC were identified. In the present UPTC 150 Cockle Northen Ireland + + study, the nucleotide positions used are for those of UPTC 182 Sea water Northen Ireland + + UPTC CF89-12 (AB736167 for sodB and AB736168 UPTC 476 Mussel Northen Ireland + + for sodC). These were predicted to encode peptides of UPTC 504 Mussel Northen Ireland + + 217 and 179 amino acid residues with calculated mo- UN C. lari JCM2530 Seagull Japan + + lecular weights (CMWs) of 24.1 and 19.9 kDa, respec- UN C. lari 28 Mussel Northen Ireland + + tively. These two possible ORFs of sodB and sodC UN C. lari 34 NA Northen Ireland + + genes were identified, based on comparison of nucleo- UN C. lari 170 Seagull Japan + + tide and deduced amino acid sequence similarities with UN C. lari 175 Seagull Japan + + those of the corresponding genes from UN C. lari UN C. lari 176 Black-tail gull Japan + + RM2100 (NC_012039) (sodB and sodC)and C. jejuni NCTC11168 (NC_002163) (sodB). UN C. lari 264 Mussel Northen Ireland + + UN C. lari 274 Mussel Northen Ireland + + Molecular and comparative analyses of sodB and sodC genes UN C. lari 295 Human Canada + + in other thermophilic Campylobacter organisms UN C. lari 298 Human Canada + + UN C. lari 299 Human United States + + As shown in Figs. 3 and 4, sodB and sodC genes are located UN C. lari 300 Seagull United States + + approximately 55–56 kbp from each other within genomic UN C. lari 448 Mussel Northen Ireland + + DNA in the UN C. lari RM2100 strain. In the UPTC CF89-12 UN C. lari 84C-1 Human Northen Ireland + + strain, these two genes are also located in a similar manner (data not shown). sodB and sodC genes in UPTC CF89-12 strain shared relatively high nucleotide sequence similarity (92.9 % and gene segments with the C. lari isolates (n =14 for UPTC and 91.7 %) with those in the UN C. lari RM2100 strain, respec- n =14 for UN C. lari; Table 3), as shown in Fig. 5. tively. In addition, sodB genes in UPTC CF89-12 and UN The primer pairs used generated the expected C. lari RM2100 strains shared relatively low nucleotide se- amplicons for sodB (Fig. 5a)and sodC (Fig. 5b)gene quence similarities with those in C. jejuni NCTC11168 segments with all 28 C. lari isolates, respectively. Thus, the present results indicate strongly that both the sodB (80.8 % and 81.7 %), C. coli RM2228 (82.0 % and 83.1 %) and C. upsaliensis RM3195 (75.9 % and 77.0 %), respective- and sodC genes are conserved within the C. lari iso- ly. These are shown in Table 2. lates genomic DNA, consisting of the UPTC and UN C. lari taxa (Table 3). However, no sodC was identified PCR amplification of sodB and C gene segments within the other three C. jejuni , C. coli and C. upsaliensis reference strains (data not shown). Thus, Two primer pairs [f-/r-ClsodB and f-/r-ClsodC (Figs. 1 and 2)] C. lari organisms carry sodB and sodC gene loci at were employed for PCR amplification of the sodB and sodC extremely high frequency. Ann Microbiol (2014) 64:1347–1356 1353 M 1321 4 5 6 7 8 90111213 14 M15 16 17 18 19 20 21 22 23 24 25 26 27 28 bp bMM 1321 4 5 6 7 8 90111213 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 bp Fig. 5 Agarose gel electrophoresis profiles of PCR amplicons of a sodB 100 bp DNA ladder (New England BioLabs), 1–28, refer to order in and b sodC genes segments amplified using two sets of primer pairs, f-/r- Table 3 (UPTC NCTC12892–UN C. lari 84C-1) ClsodB and f-/r-ClsodC, respectively, with C. lari isolates. Lanes: M SOD activity determination Discussion Next, SOD activities were determined in C. lari isolates, In the present study, sodB and sodC gene clusters were iden- including UPTC, and compared with those of other thermo- tified in UPTC CF89-12. The genetic loci of sodB gene clusters philic Campylobacter organisms. In Table 4,the SOD activ- and their adjacent genetic loci in UPTC CF89-12 (AB736167; ities are shown with 20 C. lari isolates (n=10 for UPTC; n= AB736168), UN C. lari RM2100 (NC_012039) and some 10 for UN C. lari isolates). other thermophilic Campylobacter strains, C. jejuni The SOD activities were also measured with the other three NCTC11168, C. coli RM2228 and C. upsaliensis RM3195, thermophilic Campylobacter organisms of C. coli (n =10), were completely different (Figs. 3, 4). Relatively high sequence C. jejuni (n =10) and C. upsaliensis (n =9) (Table 4). As shown similarities of sodB geneswereseeninthese Campylobacter in Table 4, almost all the 28 isolates out of a total of the 29 organisms, both at the nucleotide and amino acid sequence demonstrated low-SOD activity (<0.15 U/μg). Thus, the 13 levels (Table 3). In addition, C. jejuni NCTC11168, C. coli C. lari isolates (n =4 for UPTC; n =9 for UN C. lari isolates) RM2228 and C. upsaliensis RM3195 did not carry the sodC out of a total of the 20 C. lari isolates showed high-SOD gene. Although bacterial SOD may support the ability to chal- activity (Table 4). In addition, in the present study, more high- lenge the host immune system, and therefore may be consid- SOD activity was seen with the nine C. lari isolates including ered as a virulence determinant, this result suggests that not UPTC than was seen with the other three thermophilic having a sodC gene is not associated with virulence in Campylobacter and Helicobacter pylori organisms (Table 4). Campylobacter organisms. 1354 Ann Microbiol (2014) 64:1347–1356 Table 4 Superoxide dismutase (SOD) activity determination of C. lari Table 4 (continued) and other bacterial isolates. C, Campylobacter; H, Helicobacter; H,high Isolate SOD activity Remarks activity; M,moderate activity; L, low activity (U/μgprotein) Isolate SOD activity Remarks (U/μgprotein) C. upsaliensis 29-3 0.0171±0.0008 L C. upsaliensis 13-1 0.0711±0.0022 L C. lari UPTC NCTC12892 0.1394±0.0422 L C. upsaliensis 21-1 0.1233±0.0038 L C. lari UPTC NCTC12894 0.1401±0.0051 L C. upsaliensis faline 104-1 0.0817±0.0057 L C. lari UPTC CF89-12 0.3931±0.0139 H C. upsaliensis faline 37-1 0.1396±0.0250 L C. lari UPTC A1 0.0684±0.0046 L C. upsaliensis LMG8850 0.0714±0.0049 L C. lari UPTC 89049 0.3226±0.0520 H C. upsaliensis G1 0.2226±0.0731 H C. lari UPTC 92251 0.2921±0.0239 H H. pylori KMT52 0.326±0.021 H C. lari UPTC 27 0.2221±0.0229 H H. pylori KMT97 0.199±0.003 M C. lari UPTC 88 0.0694±0.0097 L H. pylori NY14 0.031±0.006 L C. lari UPTC 136 0.1599±0.0236 M C. lari UPTC 150 0.1759±0.0009 M Morishita et al. (2012) C. lari JCM2530 0.1216±0.0292 L C. lari 28 0.4637±0.0084 H C. lari 170 0.4560±0.0118 H Probable ribosome-binding (RB) sites (Shine-Dalgarno se- C. lari 176 0.3917±0.0076 H quences; Benjamin 2000) that are complementary to a highly C. lari 264 0.3677±0.0405 H conserved sequence of CCUCCU close to the 3′-end of 16S C. lari 274 0.3463±0.0224 H rRNA, AGGAGA (np 6,889–6,894 bp) for sodB and AGGA C. lari 295 0.4492±0.0111 H GA (np 1,871–1,876 bp) for sodC were identified in UPTC C. lari 298 0.2737±0.0121 H CF89-12 (Fig. 6). Two putative promoters, consisting of con- C. lari 299 0.3931±0.0055 H sensus sequences at the −35 (TTGAAA; np 6,823–6,828 bp) C. lari 84C-1 0.4638±0.0006 H and −10 (TATAAA; np 6,875–6,880 bp)-like structures, were C. coli 13 0.0136±0.0010 L also identified immediately upstream of the sodB gene in C. coli 24 (DO24) 0.0592±0.0000 L UPTC CF89-12 (Fig. 6). −35 (TTGACC; np 1,780– C. coli 27 0.0557±0.0047 L 1,785 bp) and −10 (TATTAT; np 1,838–1,843 bp)-like pro- C. coli 48 (PG48) 0.0815±0.0082 L moters also occurred upstream of sodC. Regarding the sodC C. coli 110 0.0634±0.0119 L gene in UPTC CF89-12, a putative intrinsic ρ-independent C. coli 154 0.0424±0.0018 L transcriptional terminator occurred immediately downstream C. coli JCM2529 0.0574±0.0014 L of the stop codon for the sodC gene. C. coli PG20 0.0325±0.0026 L The putative anaerobic ribonucleoside triphosphate C. coli 165 0.0691±0.0023 L reductase activating protein (No. 3 in Fig. 3a,c)gene C. coli 23 (DO23) 0.0752±0.0008 L (np 7,456–8,508 bp), immediately downstream of the C. jejuni 81-176 0.0753±0.0020 L sodB gene, was identified with the possible ORF (np C. jejuni LCDC4483 0.1041±0.0011 L 7,456–8,505 bp) in UPTC CF89-12. The probable RB C. jejuni HP5090 0.0391±0.0044 L site AGGAAA (np 7,446–7,451 bp) for the putative C. jejuni HP5095 0.0592±0.0001 L protein gene was also present. The putative anaerobic ribonucleoside triphosphate reductase gene (No. 2 gene C. jejuni D3083 0.0652±0.0028 L C. jejuni ST23 0.0845±0.0065 L in Fig. 3a) (np 8,510–9,025 bp) also occurred down- stream of theputativegene(No.3gene in Fig. 3a). In C. jejuni LMG6444 0.0478±0.0017 L addition, the putative conserved hypothetical protein C. jejuni HP5084 0.0573±0.0152 L (DUF 285 domain protein) gene (No. 1 gene in C. jejuni 81116 0.0799±0.0046 L Fig. 3a) (np 9,022–9,417 bp) also occurred downstream C. jejuni 79AH88-88 0.0733±0.0000 L of the putative gene. Two RB sites, AGGA (np 8,500– C. upsaliensis Maliryn 0.0108±0.0046 L 8,503 bp) and AAGAG (np 9,006–9,010 bp) were also C. upsaliensis 41-2 0.0411±0.0042 L identified for these hypothetical and putative genes. Ann Microbiol (2014) 64:1347–1356 1355 T-rich region -10 like region RBS Start codon -AAAATAAAGACTCATTTAGTCTTTTTTAATTTAACTATGTTAGAATGCTATAAAATTTAAAAAAGGAGAACAAAATGTTT C. lari RM2100 50,266 50,345 A..........G.TG.................................T..........-..............T...... UCPT CF89-12 3,701 3,780 ********** * ********************************* ********** ************** ****** T-rich region -10 like region RBS Start codon C. lari RM2100 106,350 TTTTATAAGTTTTTTGATTATTATATTTTATAAAAAATAATTTTTCATTAATAATATATGTTACAATAAAATAA-TTTTAACAAAAGGAGAAAAAATGAAA 103,251 -............................................T.......T....................T................G......... UCPT CF89-12 1,788 1,887 ******************************************** ******* ******************** **************** ********* c d TT TT GT GC T T AT TA TA CG TA GC CG ACT AAAGCT TT AGT AT TA 2,436 2,447 AA AAATCGTTTTACTC 4,438 4,455 Fig. 6 Nucleotide sequence alignment analyses of the putative promoter genes from UPTC CF89-12 and C. lari RM2100. Nucleotide sequences structures, consisting of T-rich and −10 regions, as well as the ribosome were also examined for the transcriptional terminator structures for c binding site (RBS) and the start codon ATG, for a sodB and b sodC sodB and d sodC. For the others, refer to the legend to Fig. 3 Previously, Kikuchi and Suzuki (1984) found very Overall, C. lari organisms carry both sodB and sodC genes high SOD activities in Campylobacter strains, especially within their genome and may have higher SOD activities than in C. fetus subsp. fetus compared with those in the other three thermophilic Campylobacter and H. pylori Escherichia coli , Propionibacterium acnes and organisms (Morishita et al. 2012), which carry only one Veillonella alcalescens and Pesci et al. (1994)suggested sodB gene but no sodC. Therefore, C. lari may have an a potential role for sodB in C. jejuni intracellular sur- advantage over other thermophilic Campylobacter organisms vival. In addition, SOD is an important determinant in for survival strategies within their host environment. the ability of C. coli to survive aerobically and for optimal colonization within the chicken gut (Purdy Acknowledgments This research was supported by partially a project grant funded by a Grant-in-Aid for Scientific Research (C) (20580346) et al. 1999); however, no SOD activity studies on C. from the Ministry of Education, Culture, Sports, Science and Technology lari organisms, specific for genetically variable and di- of Japan (to M.M.). M.M. and J.E.M. were funded through a Great Britain verse species (Miller et al. 2008), have appeared in the Sasakawa Foundation (Butterfield) Award to jointly examine the clinical literature. significance of Campylobacter infection in the UK and Japan. Recently, Morishita et al. 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Published: Dec 12, 2013

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